Obtainment of estrogenic substances



' so invention: stallions, pregnant mares, and pregnant women, 30

' ride. laye c ntainin he des ed ho m ne. in stances from body-fluid sources thereof or the 45 gs constant stirring to a temperaturecd v88-40 c. 5. The process for otaining subnscr p ion z,19t,295'

UNITED STATES PATENT oFFicE.

2,1983! onrnmiimsr g gggmomc' sun- Carl W. Ebericin, Highland M N. 1., arsignor to E. R. Squibb a Sons, New York, NrYn' a corporation oi New York no Drawing. Application April 15, ms,

Serial No. 202.229

-6 Claims- (Cl. 101-14 This invention relates to the obtainment and maintained at this temperature with occaestrogenic substances from pregnancy urine and sional stirring f r h Period of 34 1 en yother body-fluid sources, and has ior its object drochloric acid is added to adjust the acidity to the provision of an extraction process in which pH 1.0, the mixture is boiled with continual stir- 5 both the yield and the purity of the extracted ring for a half hour, andcoo ed in no more than c estrogenic substances are improved. 1 2 hours; 1000' gallons of ethylene dichloride is Essentially, the process or this invention comadded, the mixture stirred to produce an emulprlses digesting a body-fluid source oi estrogenie sion, and allowed to stand and settle until two substances with a proteolytic enzyme, extracting distinct layers are produced, the estrogenic sub-. the estrogenic substance with a solvent therefor, s ances being contained\ in the w r, ethylene 10 removing the solvent, and purifying the extracted d ch o ide, la e which is sepa a a d c e estrogenic substance. trated by evaporation oi the ethylene dichloride, More particularly, the pH, of the body-fluid leaving the hormone in a condition in which it is source (e. g., fetal fluid, or the urine or stallions, easily purified y ny the known methodi- 16 pregnant mares, or pregnant women) is ad- The invention may be variously o h rw m- 1 J'usted to the optimum for thespeciiic'proteolytic bodied within the scope f the pp aim enzyme to be used, the enzyme is added, and the I claim: mixture-digested until all of the complex coml. The process for obtaining estrogenic subponents of the fluid have been broken down; stances from body-fluid sources thereof or the go then the pH is adjusted to about 1.0 by adding a group consisting f fetal d, and the urine of i0 mineral acid, preferably hydrochloric acid, and stallions, pregnant mares, and'pregnant women, the mixture is boiled for a short time, cooled which comprises digesting the source material quickly, and emulsified with anorganic waterwith a proteolytic. enzyme, and extracting the' immiscible solvent for estrogenic substances, pret-' estrogenic substance from the digested material i erably ethylene dichloride; the resulting extract with an organic'wh yw n or 86 is then separated, the solvent removed by evapestrogenic substance. oration, and the estrogenic substance urines in The p s for btaining estroscnic subs the conventional manner. stances from body-fluid sources thereoi oi the The following examples are illustrative oi the group consisting of fetal fluid, and the urine of Example 1. which comprises digesting the source material 500 gallons of pregnant. mares urine is adwith a pmtemyuc and extracting the justed by means hydrochlori acid m pH 25, estrogenic substance from the digested mate'- end 00 rams f p psin is added, the material 1 with ethylene dichmrlde- 1 is stirred, slowly raised to a temperature of 38-40 The Process taming estwgemc 35 C., and maintained at this temperature wit stances from body-fluid sources thereof of the caslonal stirring for period 1 4 m group consisting of fetal fluid, and the urine 0! tional hydrochloric acid is then added to bring l n pregnant mares, and p a t m n.

t pH down to the mixture is brought to a which comprises digesting the source material I boil with constant stirring ror ahair hour, and with a proteolv ic'cnzy e a a u ii- 0-. 4o

cooled in not more than 2 hours; then 1000 ga1-- and extracting'the es'trogenic substance from the ions of ethylene dichloride is added, and the mixdigested material with .an organic water-imture is emulsified by stirring vigoroualy; on standmiscible solvent for the estrogenic substance. ing, two layers form; the lower, ethylene dichlo- 4.- The process for obtaining estrogenic subso ution, is dr wn i a d e ethylene i group consisting of retai fluid, and the urine. r ride evaporated therefrom, 18am mob regnant mm and women; substance in .a state suitable-tor purinoation by which comm, mean t source material any the methm' with a proteolytic enzyme, adjusting the pH to m leomple 2 aboutLO, boiling, cooling, and extracting the 500 gallons of stallions. urine is W t estrogenic substance from the digested material pH 8.5 by means of caustic da, a d 300 m with an organic water-immiscible solvent ior 4 01 trypsin is added;' the-mixture icheated with the estrogenic substance.

stances from body-fluid sources thereof 01 the group consisting of fetal fluid, and the urine of stallions, pregnant mares, and pregnant women, which comprises adjusting the pH 0! the source material to about 8.5, digesting the source material with trypsin, and extracting the estrogenic substance from the digested material with an organic water-immiscible solvent for the estrogenic substance.

10 6. The process for obtaining estrogenic substances from body-fluid sources thereof of the group consisting of fetal fluid, and the urine of stallions, pregnant mares, and pregnant women, which comprises adjusting the pH of the source material toabout 2.5, digesting the source ma.- terial with pepsin, and extracting the estrogenic substance from the digested material with an organic water-immiscible solvent for the estrogenic substance.

CARL W. EBERLEIN. 

